Interaction of JMJD6 with single-stranded RNA

Abstract

JMJD6 is a Jumonji C domain-containing hydroxylase. JMJD6 binds α-ketoglutarate and iron and has been characterized as either a histone arginine demethylase or U2AF65 lysyl hydroxylase. Here, we describe the structures of JMJD6 with and without α-ketoglutarate, which revealed a novel substrate binding groove and two positively charged surfaces. The structures also contain a stack of aromatic residues located near the active center. The side chain of one residue within this stack assumed different conformations in the two structures. Interestingly, JMJD6 bound efficiently to single-stranded RNA, but not to single-stranded DNA, doublestranded RNA, or double-stranded DNA. These structural features and truncation analysis of JMJD6 suggest that JMJD6 may bind and modify single-stand RNA rather than the previously reported peptide substrates.RNA binding proteins | RNA modification | RNA splicing J MJD6 was first characterized as a receptor for phosphatidylserine (PSR), which facilitates the phagocytosis of dead and dying cells by macrophages and fibroblasts (1). Targeted deletion of gene encoding PSR in mice and morpholino knock-downs of PSR in zebrafish resulted in embryonic lethality, with severe defects in hematopoiesis and aberrant development of eye, brain, and heart (2-5). In contrast, knock-down of PSR expression in Caenorhabditis elegans produced only a mild phenotype (5). Somewhat surprisingly, sequence analysis suggested that JMJD6 contains a Jumonji C (JMJC) domain, which places it within a highly conserved, cupin fold-containing enzyme family (6-8). Further analysis demonstrated that the protein is localized specifically in the nucleus (7-9). Despite the significant effects of JMJD6 deficiency, knockout mice engulfed apoptotic cells normally (9). Based on these studies and additional sequence analysis, the protein was recategorized as an α-ketoglutarateand Fe 2þ -dependent hydroxylase and was named JMJD6 (10).Recent studies demonstrated that most JMJC domain-containing proteins function as histone demethylases by specifically acting on lysine residues in histone tails (11)(12)(13)(14). For example, the specific interactions between enzymes from the JMJD2 subfamily and methylated peptides have been structurally characterized (15-18). Interestingly, JMJD6 was reported to demethylate arginine residues in histone tails (10). Several laboratories including ours, however, have been unable to reproduce these results. In other studies, JMJD6 was identified as a lysine hydroxylase that specifically recognizes the protein tail of U2AF65, a mediator of RNA splicing (19).To resolve the disparate results and further elucidate the structure and functions of JMJD6, we determined X-ray crystallographic structures of the protein with and without α-ketoglutarate. To obtain these structures, JMJD6 was cocrystallized with a Fab fragment derived from a JMJD6-specific hamster monoclonal antibody. Intriguingly, the structure of JMJD6 is dramatically different from known structures of other JMJC domain superfamily proteins includ...

Clipping of arginine-methylated histone tails by JMJD5 and JMJD7

“…While many members of the JmjC protein family have been characterized biochemically, the functions of several members, including JMJD4-JMJD8 and HSPBAP1 remain uncharacterized or controversial. For example, JMJD6 has been reported variously to be an arginine demethylase (10,11), a hydroxylase (12,13), a single-stranded RNA-binding protein (14), or RNA demethylase (11). Although JMJD6 has been reported to possess histone arginine demethylase activity (10,11), we and others have been unable to replicate this result (12,(14)(15)(16)(17).…”

“…For example, JMJD6 has been reported variously to be an arginine demethylase (10,11), a hydroxylase (12,13), a single-stranded RNA-binding protein (14), or RNA demethylase (11). Although JMJD6 has been reported to possess histone arginine demethylase activity (10,11), we and others have been unable to replicate this result (12,(14)(15)(16)(17). Thus, the identities of proteins that reverse methylation of arginine residues, either through conventional demethylation or others, have not been resolved.…”

“…PRMT1 has been reported to methylate H4, while PRMT5 targets H2A, H3, and H4, and PRMT6 is specific for H3. However, in our hands PRMT1 transferred 14 C-methyl groups to both H2A and H4, PRMT5 modified H2A and H4 but not H3 and PRMT6 methylated H2A, H3, and H4 ( histones, regardless of the PRMT used to add methyl groups ( Fig. 2A, lanes c-JMJD5).…”

“…After methylation, the radioactive intensities of the 14 C-methylated bulk histones were compared with or without incubation with recombinant c-JMJD5 or c-JMJD7. Changes of 14 C signal were quantitated using phosphorimaging.…”

The Brd4 Extraterminal Domain Confers Transcription Activation Independent of pTEFb by Recruiting Multiple Proteins, Including NSD3

“…However, more recently, it was reported to be important for lysyl hydroxylation of U2AF65, a factor associated with RNA splicing (50). Crystallographic studies support the hydroxylase catalytic activity and suggest that JMJD6 binds single-stranded RNA (20,33). A role for Brd4 in splicing regulation is plausible.…”